Levels of anti-AMA1 antibody elicited with the four multi-allele vaccine formulations in rabbits.

<p>Gp 1 rabbits were immunised with seven AMA1 antigens (DiCo mix and AMA1 from FVO, HB3, 3D7 and CAMP parasite strains) in CoVaccine HT™, and the other groups were immunised with DiCo mix in CoVaccine HT™ (Gp 2), Montanide IMS (Gp 3) and Montanide ISA 51 (Gp 4) respectively. Antibody titres were determined by a standardized ELISA with DiCo 1, DiCo 2, DiCo 3, FVO, HB3, 3D7 and CAMP AMA1-coated plates. Data is presented on a Log2 scale as dotplots with a boxplot superpose indicating the lower and upper quartiles as well as the median per immunisation group. Within the same immunisation group, plotting symbols represent the antibody titre of individual rabbits on all coating/capture antigens.</p

Alignment of the protein sequences (aa25-545) of <i>Pf</i>AMA1 antigens used in this study.

<p>DiCo proteins were used to immunise rabbits and as capture antigens in ELISA. Natural allele AMA1 proteins were used to immunize rabbits, and as capture and competitor antigens in (competition) ELISAs. All proteins were produced in <i>Pichia pastoris</i> and are devoid of N-glycosylation sites. These have been replaced with amino acid residues (indicated in red) that occur in AMA1 sequences from other malarial species (N162Q, T288V, S373D, N422D, S423K, N499Q). Residue 162 is unique as it is also a polymorphic residue. Additionally, all sequences contain a point mutation at position 376 (K to R, indicated in orange). This was necessary to prevent protein cleavage by <i>P. pastoris</i> proteases.</p

Growth inhibition of <i>Plasmodium</i> parasites by antibodies elicited with the three DiCo mix vaccines.

<p>Antibodies from all immunisation groups were tested on each of six culture-adapted strains (7G8, CAMP, FCR3, HB3, L32 and NF54) of <i>P. falciparum</i>. Plots represent the mean % inhibition ± SEM for all antibody samples within an immunisation group. Blue filled circles (•) represent Gp 2 (DiCo mix in CoVaccine HT™, n = 8), green open diamonds (◊) represent Gp 3 (DiCo mix in Montanide IMS, n = 8) and red filled squares (▪) represent Gp 4 (DiCo mix in Montanide ISA 51, n = 5).</p

Mean % antibody depletion from FVO and 3D7 AMA1-coated plates.

<p>Antibody depletion after competition ELISA, values reported as mean % depletion (95%CI).</p><p>7Ag – vaccine containingDiCo mix + four AMA1 alleles from FVO, HB3, 3D7 and CAMP parasite strains.</p><p>3Ag – vaccine containing DiCo mix.</p><p>NM &DM – competitor antigen mixtures comprising natural AMA1 alleles (FVO, HB3, 3D7, CAMP) and DiCo mix, respectively.</p

Relationship between ELISA antibody titre and <i>in vitro</i> parasite growth inhibition.

<p>Association of antibody levels with <i>in vitro</i> antibody functionality for three of the four immunisation groups (Gps 2, 3 and 4) is shown for parasite strains whose AMA1 allelic antigens were available for antibody measurement. In order to obtain an optimal estimate of the association, growth inhibition data at all four antibody concentrations tested (6.0, 3.0, 1.5 and 0.75 mg/ml) for each sample were included. Plots are based on a four-parameter logistic function, and each symbol/colour represents individual rabbits in the same immunisation group.</p

Mean % antibody depletion from DiCo 1, 2 and 3-coated plates.

<p>Antibody depletion after competition ELISA, values reported as mean % depletion (95%CI).</p><p>7Ag – vaccine containing DiCo mix + four AMA1 alleles from FVO, HB3, 3D7 and CAMP parasite strains.</p><p>3Ag – vaccine containing DiCo mix.</p><p>NM &DM – competitor antigen mixtures comprising natural AMA1 alleles (FVO, HB3, 3D7, CAMP) and DiCo mix, respectively.</p

IgG antibody levels in all participants.

<p>Total and cytophilic IgG subclass (IgG1 & IgG3) levels of 273 individuals (<i>HBB</i> AS carriers excluded) with specificity for the panel of asexual blood stage antigens of <i>P. falciparum</i>. Box-whisker plots represent medians with 25^th and 75^th percentiles (boxes), and with 10^th and 90^th percentiles (whiskers), outliers as discrete dots. The prevalence of positive responders to each recombinant proteins is noticed between brackets. A positive responder was defined as an individual who had a level of specific IgG over the positivity thresholds. These positivity thresholds were determined, for total IgG, IgG1 and IgG3 to each recombinant proteins, from the mean reactivities+2 SD of 30 plasma samples from Dutch non-immune volunteers.</p

The association between parasite growth inhibitory activity of purified IgG and age.

<p>Parasite growth inhibitory (PGI) capacity of purified IgG plotted against age, with regression line fitted (using the regression fit command of STATA software). A significant difference in PGI (p = 0.03, Kruskall-Wallis test) was observed between groups segregated according to age (8–9 years vs 10–11 years vs 12–14 years).</p

Associations between IgG responses and anti-disease/anti-parasite immunity.

*<p>The effect of malariometric status on IgG responses (IgGt: total IgG), determined by ANCOVA, was adjusted for age.</p>**<p>Significantly different after adjustment for multiple tests (Bonferroni correction, threshold of significance: P = 0.002).</p>***<p>Logistic regression was applied for the analysis of associations with protection against malaria attacks, where status (UMA vs ACLP groups) was used as the dependent variable against the explanatory variables (IgG subtype responses (IgG3 to MSP2 FC27 and IgG1 to GLURP) and age) shown to be significantly associated by ANCOVA. OR values were assessed for 10 AU increased. Age was also associated with malaria protection in the analysis (p = 0.011, OR = 0.84).</p

Parasite growth inhibitory capacity of purified IgG in groups segregated according to malariometric status.

<p>Data from IgG purified from a total of 75 plasma samples is illustrated for groups of children segregated according to their status as either low (ACLP, <2500 parasites/ul) or high (ACHP, ≥2500 parasites/ul) asymptomatic parasitemia carriers, or those with one or more malaria attacks (UMA, parasitemia plus fever) during 12 months' follow-up. 15 samples (5 matched trios) were excluded due to large variations between triplicate samples (coefficient of variation >30%). Box-whisker plots represent medians with 25^th and 75^th percentiles (boxes), and with 10^th and 90^th percentiles (whiskers), outliers as discrete dots. Doted lines represent the arithmetic mean of parasite growth inhibition.</p